Analytical aspects of protein carbonyl group in oxidized canine serum.

Oxidative stress (OS) is an imbalance between oxidants and anti-oxidants and plays an important rolein the aetiology and/or the progression of several diseases. Protein carbonyl (PCO) groups are so farused as biomarkers of OS in humans (Colombo et al, 2015).The aim of our study was to investigate whether PCOs are present in canine serum and if they can bemeasured spectrophotometrically using a method not yet validated in dogs.The presence of PCO was investigated by Western blotting after separation by SDS-PAGE of serum atdifferent dilutions (either before or after oxidation with 10% cigarette smoke extract). Protein labellingwith 2,4-Dinitrophenylhydrazine (DNPH, Brady's reagent) was followed by a two-steps incubation withprimary anti-dinitrophenyl-KLH antibodies (rabbit IgG fraction) and secondary goat anti-rabbit IgG, HRPconjugate. Signal was developed with Enhanced Chemiolumionescence (ECL).Serum PCO were quantified using a commercially available assay (Protein Carbonyl Content Assay Kit -Abcam, UK), based on derivatization of proteins with 1,4-dinitrophenylhydrazine (DNPH) andsubsequent formation of protein-conjugated dinitrophenylhydrazones (DNPs) with a peak absorbanceat 366 nm.Western blot showed an evident band of apparent MW of 69 kDa, consistent with carbonylated dogserum albumin. The spectrophotometric assay failed to demonstrate any signal at 366 nm using themanufacturer’s instructions or modified protocols.This study demonstrated that PCO are present in oxidized canine serum. However, thespectrophotometric assay employed in this study is not enough sensitive to detect PCOs. Furtherstudies are needed to assess whether this depends on the poor re-solubilisation of DNPs, or on the lowconcentration of PCOs in dogs

Preliminary data about Paraoxonase-1 (PON-1) as a maker for Feline Infectious Peritonitis (FIP)

Feline infectious peritonitis (FIP) is a fatal disease in which the definitive diagnosis is achieved by immunohistochemistry (IHC) on post-mortem biopsies. The clinical suspicion is aroused by signalment, clinical signs and several laboratory tests, including alpha-1-acid glycoprotein measurement for which the only validated kit is no longer available. Paraoxonase-1 (PON-1) is a serum enzyme with antioxidant activity, considered as a negative acute phase protein in several species. Since inflammation plays a major role in FIP, and due to the high susceptibility of cats to oxidation, it could be of great interest the evaluation of this enzyme as a diagnostic marker for FIP. The aim of this study was to measure paraoxonase-1 in healthy cats and cats with clinical signs consistent with FIP (both wet or dry form), in order to evaluate the utility of this parameter in the diagnosis of FIP. Sixty-two cats were enrolled and divided into three groups: healthy (n=16), confirmed FIP (n=22) and NON FIP with similar clinical signs (n=24). PON-1 was measured on serum, using a paraoxon-based enzymatic method, already validated in cats. Results showed significantly lower PON-1 activity in FIP cats (mean ± SD: 29.1 ± 16.3 U/mL; median: 24.4; IQR: 16.6-38.3), compared with healthy cats (90.1 ± 24.1 U/mL; median: 86.0; IQR: 76.7-105.7; P<0.001) and with “non-FIP” cats (55.9 ± 28.3 U/mL; median: 51.9; IQR: 35.7-68.8, P<0.001). A significant difference was also found between healthy and “non-FIP” cats (P<0.001). The receiver operating characteristic (ROC) curve demonstrated that PON-1 may discriminate cats with and without FIP (Fig.1). At the cut-off that maximizes the diagnostic power of the test, sensitivity and specificity for FIP were 77% each, suggesting that PON-1 may be a reliable marker in association with other confirmatory tests and with signs consistent with the disease

A reverse transcription loop-mediated isothermal amplification (LAMP) assay for the detection of feline Coronavirus

Loop-mediated isothermal amplification (LAMP) is a molecular method that amplifies DNA underisothermal conditions. It relies on the use of 4 different primers recognizing 6 regions of the templatesequence and on the use of a DNA polymerase with strand displacement activity (Notomi et al., 2000).The addition of two loop primers allows the reaction time to be of one hour only (Nagamine et al., 2002).The aim of this study was to develop a reverse transcription LAMP assay for an easy and inexpensivedetection of feline Coronavirus (FCoV). Six primers binding the conserved 3’UTR region of the FCoVwere designed with the Primer Explorer software. Thirty-two samples of RNA (11 feces, 8 effusions, 9blood samples and 4 tissues) on which a reverse transcription polymerase chain reaction (RT-PCR) forthe 3’UTR region was performed were used. The reaction was carried out in 25μL reaction volume andthe mixture was incubated in a thermocycler at 63°C for 1 hour followed by 10 minutes at 80°C. LAMPproducts were visualized under UV after electrophoresis migration on a 1.5% agarose gel stained withethidium bromide, where they produce a ladder-like pattern if positive. Results where compared withthose obtained on standard PCR. Sensitivity and specificity were respectively 60% and 100% on feces,40% and 100% on effusions, 25% and 100% on blood, and 100% and 100% on tissues. The overall sensitivityand specificity of this method were of 57.1% and 100%, thus limiting a clinical application of this method,except for tissues

The gut microbiome and mucosal defenses in cats with coronaviruses: a pilot study

Feline Infectious Peritonitis (FIP) develops from a mutation of enteric feline coronaviruses (FCoVs) and an imbalance of the host immune response. The wide polymorphism of FCoVs is associated with the viral replication rate (Licitra et al. 2013).  Changes in the composition of the gut microbiota may induce quali-quantitative modifications in FCoVs and/or different immune profiles (Weese et al., 2015). Few information is available on feline gut microbiome and the association between microbiota and the predisposition to pathological conditions (Ramadan et al., 2014).The aim of this study is to provide preliminary data about the composition of gut microbiota in healthy cats compared with FCoV infected cats (with and without  FIP), in order to evaluate whether changes of gut microbiota may induce changes in FCoV, in its genetic polymorphism and in the mucosal immunity.Screening analyses have been performed on 22 cats:- Routine hematology and biochemistry on EDTA and serum (included electrophoresis and alpha-1-acid glycoprotein measurement for cats suspected with FIP)- Nested RT-PCR-3’UTR on frozen faeces- Effusion evaluation- FIV/FeLV serologyDue to strict inclusion criteria (cats younger than 2.5 years old, indoor and not assuming antibiotics in the previous two months) and based on the results obtained from the complete set of analysis, only 15 cats, specifically 5 cats for each of the following 3 groups: FIP- affected, healthy negative and positive for FCoV, have been recruited to perform the following analyses: - microbiota analysis through NGS of 16S rRNA gene (V4 region) amplicons followed by bioinformatic analysis -  evaluation of secretory IgA (ELISA kit)- phylogenetic analysis of FCoVs S gene sequencesInnovative results will be provided on the feline gut microbiota composition. These will be correlated with the presence and genetic polymorphisms of FCoV and mucosal defenses to establish significant correlations between the analysed factors

Comparison between the diagnostic accuracy of clinico-pathological and molecular tests for feline infectious peritonitis (FIP)

The aim of this study was to compare the diagnostic accuracy for feline infectious peritonitis (FIP) of conventional clinic-pathological tests with that of molecular tests such as routine PCR and PCR followed by the sequencing of the Spike (S) gene. Blood, effusion and tissues specimens were collected from 21 FIP suspected cats. In vivo examination consisted of CBC, serum protein electrophoresis, AGP measurement, cytological and biochemical examination and the evaluation of the ΔTNC on effusions, and of molecular tests such the screening PCR (target: 3’UTR region) and the PCR directed towards the S gene followed by the amplification products sequencing in order to detect the aminoacidic substitution recently considered diagnostic for FIP1. These molecular techniques were applied to tissues collected during necropsy, which also allowed forming an FIP group (13 cats) and a non-FIP group (5 cats) based on histology and immunohistochemistry. The best test on tissues was immunohistochemistry (sens: 92.3%; spec: 100%), while the screening PCR suffered of low specificity (spec: 33.3%) and the S gene sequencing showed low sensitivity (sens: 69.2%).On effusions, the best tests resulted screening PCR and cytology (sens and spec: 100%) in comparison with the ΔTNC measurement (sens: 85.7 %; spec: 100%) and the S gene sequencing (sens: 42.8%; spec: 100%).On blood, the best test resulted AGP measurement (sens: 81.8%; spec: 100%), while serum protein electrophoresis showed a surprisingly low sensitivity (sens: 41.7%). Screening PCR (sens: 55.6%; spec: 100%) and S gene sequencing (sens: 33.3%; spec: 100%) proved again low accuracy.

Diagnosis of sepsis in dogs by measuring carbonylated proteins (PCOs) and paraoxonase (PON-1)

An early diagnosis of sepsis could allow a better prognosis and avoid the abuse of antibiotic administration; unfortunately, in veterinary medicine, specific and sensitive markers of sepsis are not available.Because Protein Carbonyls (PCOs), that result from protein oxidation, are widely used in human medicine as sepsis markers , the aim of our study was to validate an ELISA kit (Enzolifesciences, 3V Chimica, Roma) on canine serum and to measure PCOs, after a preliminary validation study, in three groups (homogeneous for age and size): healthy dogs without clinical or laboratory abnormalities (A, n=14), dogs with septic (B, n=14) and non-septic inflammation (C, n=12) at the first presentation and without previous treatments. Moreover, Paraoxonase-1, a negative acute phase protein with anti-oxidant properties (PON-1) was measured in each group with a method already validated in dogs.A Kruskal-Wallis test followed by a Wilcoxon signed rank test was used to evaluate differences between groups.The ELISA method for measuring PCOs showed a very good precision (coefficient of variation <12%) and a good accuracy in spiking-recovery tests.Compared with controls, the concentration of PCOs was significantly higher either in dogs with sepsis (P<0.001) or in dogs with non-septic inflammation (P=0.005) but no significant differences were found between the two groups of sick dogs. Conversely, PON-1 was significantly lower in sick dogs compared with controls (P<0.001 for both groups) and in septic dogs compared with dogs with non-septic inflammation (P=0.001).A negative correlation between the two markers was found (P<0.001, r=-0.594) Receiver operating characteristic (ROC) curves demonstrated that both markers may discriminate dogs with sepsis with the other groups. However, PCO was less sensitive than PON-1 in diagnosing sepsis.Future studies should be focused on the association of PCOs with other inflammatory markers, as well as the possible prognostic role of PCOs based on the outcome of the enrolled patients

Validation of a Paraoxon-based method for measurement of Paraoxonase (PON-1) activity and establishment of RI in horses

Paraoxonase-1 (PON-1) is an anti-oxidant compound considered as negative acute phase protein in animals (Rossi et al., 2013) and people (Novak et al., 2010). The paraoxon-based method for measurement of PON-1 in equine serum has not yet been validated.The aim of this study is to validate a paraoxon-based method to measure PON-1 and to establish reference intervals (RIs) in healthy horses and foals.120 horses (40 geldings, 40 stallions, 40 mares; median age: 11 years; 57 Warmbloods, 46 Trotters) and 55 foals (27 females, 28 males; median age: 47 days; 22 Warmbloods, 31 Trotters) considered healthy after physical examination and biochemistry were examined. Horses were grouped by breed: Thoroughbreds, Trotters, Warmbloods, Draft horses and Ponies. Serum PON-1 was measured with an automated spectrophotometer and an enzymatic method validated in other species (Giordano et al., 2013). After the analytical validation (precision, accuracy, interference studies), RIs were determined using the Reference Value Advisor software, according to ASCVP guidelines (Friedrichs et al., 2012). The possible gender-, age- and breed-related differences were statistically investigated.The paraoxon-based method was precise (CVs <4.0%) and accurate (P<0.001 in linearity under dilution and spike-recovery testing) but is affected by interference from mild bilirubinemia, severe lipemia or hemoglobinemia. The RIs recorded in the whole population was 38.1-80.8 U/mL. According to the Harris and Boyd test, separate RIs are recommended only for adult females and for Warmblood and Trotter adults (Figure 1).This study demonstrated that analytical performances of the paraoxon-based method for measurement of PON-1 in horses are acceptable. PON-1 is lower in horses than in other species.If future studies will demonstrate that oxidative stress induces a significant decrease of PON-1, this results will be useful to correctly classify healthy and sick horses; PON-1 could be used, as in human medicine, as a marker of oxidative stress

Effect of domperidone (leisguard®) on antibody titers, inflammatory markers and creatinine in dogs with leishmaniosis and chronic kidney disease

Altres ajuts: Ecuphar Italia IT0189/2018-PRVPRO-IP00Immunotherapeutic drugs, such as domperidone, have been shown to be promising treatments against canine leishmaniosis (CanL), but limited data are available. The aim of this pilot study (therapeutic, prospective and non-controlled) was to evaluate the effect of domperidone on serum antibody titers of Leishmania infantum, globulins, gamma globulins, acute-phase proteins (e.g. C-reactive protein [CRP]), big endothelin-1 (big ET-1), serum creatinine (SC) and proteinuria in dogs with leishmaniosis affected by chronic kidney disease (CKD). Dogs were recruited if "exposed" to or "infected" with L. infantum and affected by CKD (IRIS stage 1 [proteinuric] or IRIS stage 2-3a [SC < 3.5 mg/dl; proteinuric or non-proteinuric]). After inclusion, an oral suspension of domperidone was administered, and the dogs were followed up for 180 days, with checks at 30, 60, 90 and 180 days after initial treatment. Of the 14 recruited dogs, nine showed a statistically significant reduction in SC (χ 2 = 9.1, df = 3, P = 0.028), but not in the urine protein/creatinine ratio (χ 2 = 6.43, df = 3, P = 0.092). All dogs showed a significant reduction in antibody titers for L. infantum (χ 2 = 9.56, df = 2, P = 0.008), globulins (χ 2 = 11.08, df = 3, P = 0.011) and gamma globulins (χ 2 = 12.38, df = 3, P = 0.006) during the study period. There was also a statistically significant reduction in CRP (χ 2 = 16.7, df = 3, P = 0.001), but not in big ET-1 (χ 2 = 2.04, df = 3, P = 0.563). This study provides preliminary results on the ability of domperidone to improve SC and reduce anti- L. infantum antibody titers, globulins, gamma globulins and CRP in dogs with leishmaniosis and CKD

Haematological and biochemical reference intervals in healthy racing and retired Italian Greyhounds

In view of the enormous variability of dog breeds, breed-specific reference intervals (RIs) are recommended for use in veterinary clinical decision-making. The aim of this study was to determine whether RIs of the general canine population may be applied to the Italian Greyhound (Piccoli Levrieri Italiani or PLI), and to generate breed-specific RIs, where appropriate. Sixty-three privately owned clinically healthy fasted dogs were examined. Routine haematology and biochemistry were performed on 58 enrolled patients using the ADVIA 120 haematology analyzer and the Cobas Mira system, respectively. Changes in haematological and biochemical parameters depending on sex, age and attitude (resting vs. running dogs) were investigated. The results of PLI were compared with the RIs of the general canine population. In those cases in which these RIs were not validated, new RIs were generated according to the guidelines of the American Society of Veterinary Clinical Pathology. Pre-existing RIs were considered valid based on the recommendations by the Clinical &amp; Laboratory Standards Institute (CLSI). RIs were higher for mean corpuscular haemoglobin (MCH), mean cell haemoglobin concentration (MCHC), cell haemoglobin concentration mean (CHCM) and lower for large unstained cells (LUC). A wider discrepancy between pre-existing and newly established RIs was found for some ADVIA parameters regarding red blood cell (RBC) or reticulocyte morphology. For total protein and cholesterol the new RIs were wider than the pre-existing ones, while albumin, calcium and iron were higher. This study suggests that most of the RIs published in veterinary textbooks cannot be validated for PLIs